Enzyme substrate delivery and product registration in one step enzyme immunoassays

ABSTRACT

One-step enzyme immunoassays in which enzyme-antibody conjugate or label and enzyme substrate are separated until separation of bound and free enzyme conjugate or label is complete. This separation is accomplished by using variable flow paths, immobilization of substrate at the test line, placement of substrate in a sac or association with a particle label, enzyme product chemical capture, delay zone dissolution and protected enzyme substrates.

This application is a divisional of prior application Ser. No.08/977,183, filed on Nov. 24, 1997, now U.S. Pat. No. 6,306,642, fromwhich priority under 35 U.S.C. §120 is claimed.

FIELD OF THE INVENTION

The present invention relates to the detection of analytes in biologicalfluids. More specifically, the invention relates to enzyme substratedelivery and product registration in one step enzyme immunoassays.

BACKGROUND OF THE INVENTION

Analyte-specific binding assays are important tools for detecting andmeasuring environmental and biologically relevant compounds, includinghormones, metabolites, toxins and pathogen-derived antigens. Aconvenient version of the binding assay is an immunoassay which can beconducted in a “lateral flow” format.

Devices useful for performing lateral flow assays typically includeseveral “zones” that are defined along a length of a matrix. The matrixdefines a flow path and provides fluid connection between the variouszones, including a sample receiving zone, a labeling zone forspecifically labeling the analyte, and a capture (detection) zonelocated downstream from the sample receiving zone and the labeling zone.An absorbent zone (sink) typically is located downstream of the capturezone, and provides a means for removing excess sample and unbound labelfrom the matrix.

In some applications the matrix of a lateral flow assay device is amembrane capable of “non-bibulous lateral flow.” In these applicationsliquid flow occurs such that all of the dissolved or dispersedcomponents in the analyte-containing liquid are carried at substantiallyequal rates and with relatively unimpaired flow laterally through themembrane. This is distinguished from a situation wherein preferentialretention of one or more components occurs, for example, in materialscapable of adsorbing or imbibing one or more of the components.

A principal advantage of the lateral flow immunoassay is the ease withwhich the testing procedure is carried out. In this procedure a fluidsample first contacts the matrix following application to the samplereceiving zone. Capillary action then draws the liquid sample downstreaminto a labeling zone that contains a means for indirectly labeling thetarget analyte. The labeling means generally will be a labeledimmunoglobulin, but alternatively may be a non-immunoglobulin labeledcompound which specifically binds the target analyte. After flowingthrough the labeling zone, the sample continues to flow into the capturezone where it contacts an immobilized compound capable of specificallybinding the labeled target analyte or the complex formed by the analyteand label. As a specific example, analyte-specific immunoglobulins canbe immobilized in the capture zone. Labeled target analytes will bindthe immobilized immunoglobulins upon entering the capture zone and willbe retained therein. The presence of the labeled analyte in the sampletypically will be determined by visual detection of the label within thecapture zone. Finally, the procedure is complete when excess sample istaken up by the material of the absorbent zone.

Lateral flow immunoassays typically employ test and procedural controllines in the capture zone. The test line serves to detect an analytepresent in a test sample, while the procedural control lineconventionally serves to detect a ligand unrelated to the analyte.Rather than being applied in the test sample, the ligand unrelated tothe analyte is disposed in the labeling zone of the lateral flowimmunoassay device. The test line ordinarily employs specificcompetitive, sandwich or indirect binding separation principles using avisual label. This requires the use of a labeled detector antibody inthe labeling pad of the labeling zone and a capture antibody or ligandimmobilized at the capture test line.

The capture zone of lateral flow immunoassay devices may also include aprocedure control line useful for indicating that a procedure has beenperformed. The procedure control line generally is located downstream ofthe binding compound that is immobilized in the capture zone at the testline where reaction occurs. Retention of label by the procedural controlline indicates that liquid sample has flowed through the capture zoneand contacted the immobilized target-specific binding substance. Theaccumulation of visible label may be assessed either visually or byoptical detection devices.

Another type of enzyme immunoassay utilizes a flow-through device whichis described in U.S. Pat. No. 4,632,901. This device comprises amembrane or filter to which an antibody is bound. An absorbent materialin contact with the membrane or filter induces flow therethrough when afluid sample is added to the membrane or filter. A fluid sample isapplied to the membrane and, if the cognate antigen is present, is boundby the antibody. A solution of labeled antibody against the antigen isthen added followed by a washing step to remove unbound labeledantibody. The presence of labeled antibody on the membrane after washingindicates the presence of the antigen in the sample being assayed.

In one step enzyme immunoassays (EIAs), whether they be flow-through orlateral flow constructs, there is an inherent limitation to the use ofenzyme amplification wherein the enzyme (as either enzyme-antibodyconjugate or enzyme-label particulate) must be kept separate from itssubstrate until separation of bound and free enzyme conjugate or labelis complete. The present invention addresses methods for suchseparation.

SUMMARY OF THE INVENTION

One embodiment of the present invention is an enzyme immunoassay device,comprising a sample pad comprising a slow lane and a fast lane separatedby a hydrophobic barrier, wherein the slow lane contains an enzymesubstrate and the fast lane contains an enzyme-antibody conjugate havingaffinity for an analyte; a capture zone in fluid communication with thesample pad, the capture zone having a capture antibody incorporatedtherein having affinity for said analyte; and an absorbent zone in fluidcommunication with said capture zone. Preferably, the analyte is ahormone, enzyme, lipoprotein, bacterial antigen, viral antigen,immunoglobulin, lymphokine, cytokine, drug or soluble cancer antigen.Advantageously, the sample pad comprises high density polyethylene.

Another embodiment of the present invention is a flow-through lateralflow enzyme immunoassay device, comprising a disk comprising inner andouter hydrophilic zones separated by a hydrophobic barrier, the innerzone containing an enzyme substrate and having a smaller pore size thanthe outer zone, the outer zone containing an enzyme-antibody conjugatehaving affinity for an analyte; a contact pad in fluid communicationwith the molded disk; a capture zone in fluid communication with thecontact pad; and an adsorbent zone in fluid communication with thecontact pad.

The present invention also provides a lateral flow enzyme immunoassaydevice, comprising a sample pad; a label pad in fluid communication withthe sample pad, the label pad containing an enzyme-antibody conjugatehaving affinity for an analyte; a capture zone in fluid communicationwith the label pad, the capture zone containing a capture antibodyhaving affinity for the analyte; and an enzyme substrate at a test line.In one aspect of this preferred embodiment, the substrate is chemicallyimmobilized at the test line. Alternatively, the substrate isimmobilized in a mordant under the test line. Still alternatively, thesubstrate is immobilized in a mordant dispensed within the test line.Preferably, the capture zone further comprises chemical groupsincorporated therein, the chemical groups capable of specificallyreacting with the product resulting from enzyme action on the substrate.Advantageously, the chemical groups comprise diazotized amines.

Another embodiment of the invention is a lateral flow enzyme immunoassaydevice, comprising a sample pad; a label pad in fluid communication withthe sample pad, the label pad containing a substrate covalently attachedto a particle or imbibed within a sac, wherein the substrate-containingsac or particle is attached to an antibody; a capture zone in fluidcommunication with the label pad, the capture zone containing anenzyme/mediator for releasing the substrate and a capture antibody at atest line; and an absorbent zone in fluid communication with the capturezone. Preferably, the sac comprises a liposome. Alternatively, the saccomprises an erythrocyte ghost. Advantageously, the particle labelcomprises polyalkylcyanoacrylate polymer monosized colloids. Theenzyme/mediator may immobilized in a mordant within or under the testline, or may be attached to the capture antibody.

The present invention also provides an enzyme immunoassay device,comprising a sample pad comprising a first lane containing a firstbarrier zone and a second lane containing a second barrier zone, whereinthe first lane contains an enzyme-antibody conjugate having affinity foran analyte and said second lane contains an enzyme substrate, whereinthe first barrier zone dissolves before said second barrier zone; acapture zone in fluid communication with the sample pad, the capturezone containing a capture antibody incorporated therein having affinityfor the analyte; and an absorbent zone in fluid communication with thecapture zone. The barrier zones may comprise structural hydrogel,enterosoluble coatings or biodegradable phospholipids.

Still another embodiment of the invention is an enzyme immunoassaydevice, comprising a sample pad containing a first enzyme and a secondenzyme, the second enzyme conjugated to a second antibody havingaffinity for an analyte; a label pad in fluid communication with thesample pad, the label pad containing a substrate for the first enzyme,wherein the substrate for the first enzyme is converted by the firstenzyme to a second substrate for the second enzyme; and a capture zonein fluid communication with the label pad, the capture zone containing afirst antibody having affinity for the analyte at a test line, whereinthe second substrate is converted by the second antibody to an enzymeproduct; and an absorbent zone in fluid communication with the capturezone. Preferably, the first enzyme is alkaline phosphatase, esterase,protease, sulfatase, chymotrypsin-like protease, creatineamidinohydrolase or arginase. Advantageously, the second enzyme isβ-D-galactosidase, N-acetylglucosaminidase, α-L-arabinofuranosidase,exglucanase, chitobiosidase, α-L-fucosidase, β-D-glycosidase,α-galactosidase, β-glucosidase, glucansucrase, β-D-glucuronidase,α-amylase, α-mannosidase or β-mannosidase. According to another aspectof this preferred embodiment, the analyte is a hormone, enzyme,lipoprotein, bacterial antigen, viral antigen, immunoglobulin,lymphokine, cytokine, drug or soluble cancer antigen.

The present invention also provides a sample receiving layer for use inan enzyme immunoassay device, comprising: a disk comprising inner andouter hydrophilic zones separated by a hydrophobic barrier, the innerzone containing an enzyme substrate and having a smaller pore size thanthe outer zone, the outer zone containing an enzyme-antibody conjugatehaving affinity for an analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a lateral flow assay strip containing avariable flow path sample pad.

FIG. 2 is a schematic diagram of a lateral flow enzyme immunoassay stripin which the substrate is covalently attached to a particle label orimbibed inside a sac. X=analyte; SP=sample pad; LP=label pad;Nitro=nitrocellulose; S=substrate; Enz/Med=enzyme/mediator.

FIG. 3 is a schematic diagram of enzyme product chemical capture. Theanalyte X is bound by both an enzyme (E)-conjugated antibody and acapture antibody. The enzyme substrate (S) is converted to product (P)which binds to chemical groups (R) immobilized on the support.

FIG. 4 is a schematic diagram of an enzyme immunoassay test strip fordelayed enzyme substrate delivery using hydrogel barrier zones.

FIG. 5 is a schematic diagram of an enzyme immunoassay test strip forsequential delayed enzyme-antibody and enzyme substrate release usinghydrogel barrier zones.

FIG. 6 is a schematic diagram of an enzyme immunoassay test strip forsequential delayed enzyme-antibody and enzyme substrate release usingenterosoluble barrier zones.

FIG. 7 is a schematic diagram of delayed release of enzyme substrateusing multi-enzyme systems and protected enzyme substrates. X=analyte;E₁=enzyme 1; E₂=enzyme 2; S=substrate; P=product.

FIG. 8 is a schematic diagram of delayed release of enzyme substrateusing the β-D-galactosidase anti-analyte system.

FIG. 9 shows alternative “protected” substrates for theβ-D-galactosidase-Ab conjugate shown in FIG. 8.

FIG. 10 shows “protected” substrates for urease as E1 in E1 Abconjugates and the resulting products.

FIG. 11 is a top view of a molded disk comprising variable flow pathsfor use as a top sample receiving layer in the immunoassay device shownin FIG. 12.

FIG. 12 is a cross-sectional view of a flow-through lateral flow enzymeimmunoassay device.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides one step enzyme immunoassay devices andmethods in which enzyme and substrate are kept separated untilseparation of bound from free enzyme conjugate or label is complete. Theinvented assay devices comprise four distinct zones. The device isdesigned so that the analyte-containing sample is first applied to asample receiving zone, then flows through a labeling zone and into acapture zone. The capture zone in turn is in contact with an absorbentzone which provides a means for removing excess liquid sample. Inconventional immunoassay devices, the absorbent zone consists of anabsorbent such as filter paper or glass fiber filter.

As used herein, the term “pad” refers to the physical material whichcorresponds to a zone or section of an immunoassay strip. Thus, a samplepad is the material of the sample receiving zone of an immunoassaystrip. The labeling pad similarly refers to the material of the labelingzone.

The invention generally concerns one-step lateral flow or flow-throughassays which are conducted on supports which may conduct nonbibulouslateral flow of fluids. As defined herein “nonbibulous” lateral flowrefers to liquid flow in which all of the dissolved or dispersedcomponents of the liquid which are not permanently entrapped or“filtered out” are carried at substantially equal rates and withrelatively unimpaired flow laterally through the membrane or support.This is distinguished from preferential retention of one or morecomponents as would occur, for example, in materials capable ofabsorbing or “imbibing” one or more components as occurs inchromatographic separations. “Bibulous” materials include untreatedforms of paper, nitrocellulose and the like which effect chromatographicseparation of components contained in liquids passed therethrough.Bibulous materials can be converted to materials which exhibitnonbibulous flow characteristics by the application of blocking agents.These agents may be detergents or proteins which can obscure theinteractive forces that account of the bibulous nature of the supports.Thus, nonbibulous materials include those which are intrinsicallycapable of conducting nonbibulous flow, such as porous polyethylenesheets or other inert materials or can be comprised of bibulousmaterials which have been blocked. Preferred blocking agents includebovine serum albumin, either per se or in methylated or succinylatedform, whole animal sera, such as horse serum or fetal calf serum, andother blood proteins. Other examples of protein blocking agents includecasein and nonfat dry milk. Detergent-based blocking agents can also beused for rendering a bibulous material capable of nonbibulous flow. Thetypes of detergents appropriate for this purpose are selected fromnonionic, cationic, anionic and amphoteric forms, and the selection isbased on the nature of the membrane that is being blocked.

To convert a bibulous support such as paper or nitrocellulose to asupport capable of effecting nonbibulous lateral flow, the originalsupport is treated with a solution of the blocking agent in an effectiveconcentration to dispose of unwanted reactivities at the surface. Ingeneral, this treatment is conducted with a blocking solution, such as aprotein solution of 1-20 mg/ml protein at approximately room temperaturefor between several minutes and several hours. The resulting coatedmaterial is then permanently adsorbed to the surface by air-drying,lyophilization, or other drying methods. Both the selection andtreatment of carrier porous materials used to construct immunoassaystrips of the sort described herein depend on the functional role thateach zone performs in the assay device.

The sample-receiving zone serves to begin the flow of analyte-containingsample, and typically will be constructed of a material that exhibitslow analyte retention. One means for imparting this property involvesimpregnating the sample receiving zone with a neutral protein-blockingreagent, followed by treatment to immobilize the blocking agent (e.g.,lyophilization). An additional advantage of this treatment is increasedwetability and wicking action which speeds transfer of the liquid sampleinto the labeling zone. The sample-receiving zone may also function as amechanical filter, entrapping any undesirable particulates present inthe sample.

The labeling zone contains enzyme-antibody conjugate or particulatemoieties which may or may not be visible, which can be detected ifaccumulated in the capture zone. The visible moieties can be dyes ordyed polymers which are visible when present in sufficient quantity, orcan be, and are preferred to be particles such as dyed latex beads,liposomes, or metallic, organic, inorganic or dye solutions, dyed orcolored cells or organisms, red blood cells and the like. Theenzyme-antibody conjugate or particulate moieties used in the assayprovide the means for detection of the nature of and quantity of result,and accordingly, their localization in the capture zone must be afunction of the analyte in the sample. In general, this can beaccomplished by coupling the enzyme-antibody conjugate or particulatemoieties to a ligand which binds specifically to the analyte, or whichcompetes with analyte for a capture reagent in the capture zone. In thefirst approach, the conjugate or particulate moieties are coupled to aspecific binding partner which binds the analyte specifically. Forexample, if the analyte is an antigen, an antibody specific for thisantigen may be used; immunologically reactive fragments of the antibody,such as F(ab′)₂, Fab or Fab′ can also be used. These conjugate orparticulate moieties, or “test” moieties, then bind to analyte in thesample as the sample passes through the labeling zone and are carriedinto the capture zone by the liquid flow. When the complex reaches thecapture zone, it is captured by an analyte-specific capture reagent,such as an antibody. Excess liquid sample finally is taken up by theabsorbent zone. In the second approach, the conjugate or particulatemoieties are coupled to a ligand which is competitive with analyte for acapture reagent in the capture zone, most typically, other molecules ofthe analyte itself. Both the analyte from the sample and the competitorbound to the conjugate or particulate moieties are then carried into thecapture zone. Both analyte and its competitor then react with thecapture reagent, which in this instance is also typically specificallyreactive with an analyte and its competitor. The unlabeled analyte thusis able to reduce the quantity of competitor-conjugated conjugate orparticulate moieties which are retained in the capture zone. Thisreduction in retention of the conjugate or particulate moieties becomesa measure of the analyte in the sample after enzyme turnover.

The labeling zone of immunoassay devices of the present invention alsomay include a procedural control which comprises visible moieties thatdo not contain the specific binding agent or analyte competitor and thatare also carried through to a control area of the capture zone by theliquid flow. These visible moieties are coupled to a control reagentwhich binds to a specific capture partner and can then be captured in aseparate procedural control portion of the capture zone to verify thatthe flow of liquid is as-expected. The visible moieties used in theprocedural control may be the same or different color than those usedfor the test moieties. If different colors are used, ease of reading theresults is enhanced.

The experimental results of a procedure conducted using an immunoassaystrip are read in the capture zone by noting the presence or absence ofa visible signal at the location of the capture zone for the test line.The use of a procedural control region is helpful for indicating thetime when test results can be read. Thus, when the expected colorappears in the procedural control region, the presence or absence of acolor in the test region can be noted. The use of different colors fortest and control regions aids in this process.

The use of a matrix which is bibulous inherently, but convertible to anonbibulous flow characteristic, is particularly useful in the creationof the capture zone. Capture reagents can be applied to the matrixbefore the application of blocking agents and can be immobilized insitu. At this stage, the bibulous nature of the matrix during thecoupling of the capture reagents may be advantageous. However, theblocking/washing treatment which converts the bibulous membrane tononbibulous support provides unimpaired and speedy flow of allcomponents of the system.

The extremely rapid nature of the assay, which typically yields a resultin less than one minute, provides in many instances essentially aninstantaneous result as the sample flows due to the nonbibulous natureof the zones and of the short distance the sample must traverse in eachzone. Another factor which contributes to the speed of the assay is theabsorptive potential of the material used to create the absorbent zoneand the use of dessicant as adsorbent.

Miniaturization of the diagnostic device also contributes to theremarkable speed of the assay. Miniaturization permits instantaneousresults which are observable as soon as the sample contacts the capturezone and which occur almost immediately or within 60 seconds of theaddition of the sample to the sample receiving zone. The speed ofappearance and intensity of the positive visible reaction seen dependson the concentration of analyte in the sample. The speed of appearanceof the positive visual reaction can be adjusted to provide the optimalvisual result with concentrations of analyte of clinical importance andadjusted to suit the timing needs of the end-user.

Suitable analytes detectable by the invented immunoassay devices are anyfor which a specific binding partner can be found. In general, mostanalytes of medical and biological significance can find specificbinding partners in antibodies prepared against them or fragments ofthese antibodies. Suitable analytes include soluble analytes such ashormones, enzymes, lipoproteins, bacterial or viral antigens,immunoglobulins, lymphokines, cytokines, drugs, soluble cancer antigens,and the like. Also included as suitable analytes are hormones such ashuman chorionic gonadotropin (hCG), insulin, glucagon, relaxin,thyrotropin, somatotropin, gonadotropin, follicle-stimulating hormone,gastrin, bradykinin, vasopressin, and various releasing factors. A widerange of antigenic polysaccharides can also be determined such as thosefrom Chlamydia, Neisseria gonorrheae, Pasteurella pestis, Shigelladysentereae, and certain fungi such as Mycosporum and Aspergillus.Another major group comprises oligonucleotide sequences which reactspecifically with other oligonucleotides or protein targets. Anextensive list of soluble analytes determinable in the method of theinvention is found in U.S. Pat. No. 3,996,345, which is incorporatedherein by reference.

The invented immunoassay strips can be disposed within a housing that isboth protective and functional. In one preferred embodiment the housingis adapted to have at least one port for receiving a liquid sample andguiding fluid flow of the sample to contact the immunoassay strip at thesample receiving zone. The housing also can have windows which allow auser to view portions of the immunoassay strip, including portions ofthe capture zone and/or the absorbent zone.

Various enzyme substrate systems can be used in the one-step enzymeimmunoassays of the invention. These systems are summarized in Table 1but not limited thereto.

TABLE 1 1) SOLUBLE 2) INSOLUBLE ENZYME PRODUCT PRODUCT pH RANGE Alkaline(p-Nitrophenyl BCIP, 3-IP 9-10.5 Phosphatase phosphate) Azonaphtol(p-Aminophyenyl phosphate phosphate) BCIP/tetrazolium Azonaphthol saltphosphate 3-IP/tetrazolium salt Naphthyl phosphate Horseradish DAB/MBTY3-AEC 4-8 peroxidase ABTS 4-Chloronaphthol TMB 4-Chloronaphthol/ MBTH4-Chloronapthol/ 4-AAP DAB Glucose Tetrazolium Tetrazolium salt/ 6-8oxidase salt/PMS PMS Diaphorase Tetrazolium salt/ Tetrazolium salt/ 6-8oxidoreductase NADH (NADPH) PMS Galactosidase (Azonapthol (Indolyl 6-8galactopyranoside) galactopyranoside (Aminophenyl (Azonaphtholgalactopyranoside) galactopyranoside) (Napthyl galactopyranoside)(Nitrophenyl galactopyranoside) Urease Urea 6-8 Urea/pH indicatorGlucuronidase (Azonaphtol (Azonaphthol 6-8 glucuronide) glucuronide)(Nitrophenyl (Indolyl glucuronide) glucuronide) Naphthyl glucuronide(Aminophenyl glucuronide) 1) Product of Reaction of enzyme on substrateis water soluble. 2) Product of Reaction of enzyme on substrate is waterinsoluble.

Various embodiments of enzyme-substrate delivery and productregistration in one-step EIAs are discussed below.

1. Variable Flow Path

A lateral flow EIA device 2 is illustrated in FIG. 1. Enzyme-antibodyconjugate (E-Ab) 4 is lyophilized and placed in the fast chromatographiclane 6 of a bi-phase solid support 8. The enzyme substrate 10 islyophilized and placed in the slow chromatographic lane 12. The “fast”and “slow” lane designations refer to the pore size of the high densitypolyethylene. The slow lane contains smaller pore sizes than the fastlane. Accordingly, the substrate 10 will move through the sample padmore slowly than E-Ab 4. A volume of sample containing analyte isapplied to solid support 8 and contacts both substrate 10 and E-Ab 4simultaneously. The substrate 8 and E-Ab 4 then flow at variable ratesto the nitrocellulose detection zone 14 for sequential conjugate, thensubstrate, reaction. An immunological separation occurs in the detectionzone 14 where analyte-Ab-E complex either binds to the capture antibodyat the test line 16 or Ab-E passes unbound to the absorbent zone 18.Upon reaching the immobilized E-Ab, substrate is converted to productwhich is either insoluble and precipitates at the test line 16, or thesoluble product is measured downstream by conventional methods. Aspecific example of this embodiment is provided in Example 1.

EXAMPLE 1 Substrate and Enzyme-Antibody Conjugate Delivery UsingVariable Flow Paths

Molded hydrophilic high density polyethylene (HDPE) macroporous supportssuch as those custom manufactured by Interflo Technologies (Brooklyn,N.Y.) were used to form the variable flow paths. Two pieces of 2 mmthick hydrophilic HDPE (5 mm×50 cm each) of different pore sizes (5-20μm and 20-80 μm) were separated by a hydrophobic non-porous material (2mm×50 cm), aligned along the length parallel to each other on the sameplane, then fused together. The resultant “two-lane” strips were cutinto 12×20 mm pieces. To each two-lane strip was added a 5 μl aliquot ofenzyme-antibody conjugate (3-6 mg/ml), spotted in the center of the fastlane, and 5 μl aliquots of the appropriate enzyme substrate (5-12 mg/ml)supplemented with 10-20% (w/v) of cyclodextrin were spotted in thecenter of the slow lane. Cyclodextrin was added to increase theviscosity and further retard movement of the substrate through thestrip. The prepared materials were lyophilized and assembled asintermediate zones into a lateral “one-step” device. Subsequently,substrate and enzyme-antibody conjugate delivery by use of a variableflow path were studied by applying buffer solutions at different pH.

In a related embodiment, substrate and enzyme-antibody conjugate weredelivered using a variable flow path in a flow-through lateral flowdevice. Referring to FIG. 11, a 20 mm diameter molded disk of HDPE 100was constructed by fusing a 9 mm diameter inner disk 102 of 5-20 μm poresize hydrophilic HDPE with an outer 2 mm thick O-ring 104 of hydrophobicmedium and an outside 9 mm thick O-ring 106 of 20-80 μm hydrophilicHDPE. The inner disk 102 was saturated with the appropriate enzymesubstrate solution (0.2-2 mg/ml) containing 5-20% cyclodextrin and theoutside ring 106 was saturated with the corresponding enzyme-antibodyconjugate (5-25 μg/ml) supplemented with 2-10 mg/ml bovine serum albumin(BSA). The use of any desired diameter disk and O-ring thickness forforming the molded disk 100 is within the scope of the invention. Inaddition, the use of hydrophilic materials other than HDPE is alsocontempated. Such materials include, for example, polypropylene andpolyvinylchloride.

The resultant pieces were lyophilized and assembled as a samplereceiving top layer of the flow-through device 100 shown in FIG. 12. Thedevice has a top housing 111 and a bottom housing 113. Disk 100, whichfunctions as the label pad, is placed in sample well 112 in contact withabsorbent contact pad 114 which is in fluid communication with capturezone 116 and absorbent pad 118. Test line 120 containing a captureantibody is viewable through view window 122.

A fluid sample containing an antigen of interest is applied to thedevice 110 and, due to the larger pore size of O-ring 106 to which theenzyme-antibody conjugate is applied, the enzyme-antibody-antigencomplex moves into the contact pad 114 and capture zone 116 in fluidcommunication with disk 100 before the enzyme substrate, thus keepingthe enzyme and substrate separated until antigen has bound to theantibody at the test line 120. Unbound antigen and reagents flow intothe absorbent pad 118.

2. Substrate Delivery Through Immobilization at Test Line

In this lateral flow embodiment, the substrate is immobilized at thetest line chemically, in a mordant under the test line (e.g. protein,gel, etc.) or in a mordant dispensed within the test line in thenitrocellulose membrane (e.g. cyclodextrin, polyvinyl acetate, etc.). Ina lateral flow assay format, analyte reacts with enzyme-antibodyconjugate localized in a label pad, followed by complex formation in thepresence of antigen. The complex is captured by the capture antibodyimmobilized at the test line or proceeds unbound to the absorbent in theabsence of analyte. The enzyme product is either insoluble andprecipitates on the test line or is soluble and measured downstream.

3. Substrate Delivery Vehicles

In one embodiment of this lateral flow EIA device 20 (FIG. 2), thesubstrate 22 is covalently attached to a particle label, such as a latexparticle bound to indoxyl phosphate moieties via primary aminefunctionalities. Under the influence of alkaline phosphatase (EC 3.1.3.1from bovine and calf intestinal mucosa), the phosphate ester is cleavedto liberate indol-3-ol which in turn is oxidized by free oxygen insolution to blue indigo dye.

In another preferred embodiment, the substrate is imbibed within a sac24. One example of an enzymatic substrate imbibed within a sac involvesthe use of liposomes to carry enzyme substrates to the area whereenzymes are immobilized, presumably at the capture line. Techniques forincorporating substances into liposomes are well known in the art. Thistechnique requires disruption of the liposome at the appropriate time torelease the substrate for the enzyme release factor.

Alternatively, an enzyme substrate is encapsulated in a red blood cell(RBC) based on the fact that a RBC can be re-formed after osmotic lysisunder the proper conditions of osmolarity, temperature and pH to trapany solutes present at the time of reforming (D'Orazlo et al., Anal.Chem., 49:2083-2086, 1977), hereby incorporated by reference). Theresulting loaded erythrocyte ghosts are functionalized with the antigen(or antibody) to an antibody (or antigen) to be quantified. This may beaccomplished by mixing the antibody or antigen to be bound to the RBCsurface with a suspension of RBCs in the presence of tannic acid,chromium chloride or a water-soluble carbodiimide. The functionalizedRBC sacs are then used to carry enzyme substrate to the enzyme at thecapture line. Upon capture of the erythrocyte ghost, the hemolysin isbrought into close proximity and is able to break down the membranewall, liberating the enzyme substrate. Also, release can be accomplishedby a variety of means, including enzymes, surfactants, ionic strengthshifts, complement formation and opsonization. Thus, in FIG. 2, thecomponents are as follows: 24, substrate-loaded erythrocyte ghost; 22,antibody to target analyte on RBC surface; 30, second antibody toanalyte; 26, hemolysin conjugated to second antibody.

In both embodiments mentioned above, an enzyme/mediator 26 isimmobilized in a mordant within or under the test line 28, or attachedto the capture antibody 30. Analyte solution flows from the sample pad32 to the label pad 34 where the analyte binds to antibody 36.Analyte-antibody conjugate then binds to capture antibody 30 in asandwich format in the detection zone 38. A suitable enzyme/mediator(release factor) 26 such as Phospholipase C from C. perfringens (EC3.1.4.3) or a surfactant degrades or lyses the particles or sacs 24 torelease substrate 22. The sac 24 may be, for example, a liposomeprepared by procedures well known in the art, or an erythrocyte ghost asdescribed above. See, for example, U.S. Pat. No. 4,342,826, publishedPCT Application No. WO80/01515 and U.S. Pat. No. 4,703,017, all of whichare hereby incorporated by reference.

Other contemplated release factors include surfactants such asoctyl-β-D-glucopyranoside which is stored as a mordant under the captureline as opposed to attached or conjugated to the capture antibody. Analternative system, as described above, is the use of erythrocyte ghostswith enzyme substrates in which hemolysin serves as the enzyme releasefactor.

When the particle or sac containing the substrate is captured, enzymesubstrate leaches from the sac 24 or is released from the particle andenzyme product precipitates on the line or is measured downstream uponenzyme turnover. Unbound antibodies and analyte migrate to the absorbentzone 40.

EXAMPLE 2 Incorporation of Enzyme Substrate within a Biodegradable Sac

To a 500 ml round-bottom rotary evaporator flask was added, with mixing:240 mg cholesterol, 520 mg distearoyl phosphatidylcholine (20 mg/ml inCHCl₃), 18.75 mg distearoyl phosphatidylethanolamine-(p-maleimidophenyl)butyrate (2 mg/ml in CHCl₃); 30 mg of isopropyl ether and 5 ml methanol.Subsequently, 25-100 ml of suitable enzyme substrate (2-50 mg/ml in 0.1M sodium acetate, 0.1 M NaCl, pH 4.5) was added. The mixture was mixed,emulsified by sonication and rotary evaporated at 45-55° C. The warmliposomes were extruded sequentially through 1.0 μm, 0.7 μm, 0.5 μm,then 0.3 μm, then 0.2 μm nucleopore polycarbonate membranes. Following aseries of high-speed centrifugation (>50,000×g for>30 minutes) and washsteps (0.1 M sodium acetate buffer, 0.1 M NaCl buffer, pH 4.5),liposomes containing entrapped enzyme substrates were resuspended in 10mM Tris NaCl/EDTA buffer (pH 7.5-8.0 storage buffer) of osmolarityranging from 200-400 mOs/kg.

Subsequently, anti-human chorionic gonadotropin (hCG) monoclonalantibody was derivatized using the SPDP/DTT procedure. This methodinvolves addition of a pyridyl disulfide group to the anti hCG Mab whichis to be added to the surface of the liposome (Wong, S., CRC Chemistryof Protein Conjugation and Cross-Linking, CRC Press, Inc., 1991;Liposomes: A Practical Approach, R.R.C. New, Ed., IRL Press, 1990, bothhereby incorporated by reference.). Briefly, the antibody (about 6mg/ml) was incubated with a 50-fold molar excess of(N-succinimidyl-3)-[2-pyridyldithio]propionate) (SPDP) previouslydissolved in ethanol in 0,1 M sodium phosphate buffer, pH 7.5, for 30minutes at 25° C. Pyridyl disulfide-labeled antibody was isolated by gelfiltration through a Sephadex G-25 column. The pyridyl dithio-Mabsolution was titrated in citrate buffer to pH 5.5 by addition of 1 MHCl. a solution of 2.5 m dithiothreitol (DTT, 380 mg/ml) in 0.2 Macetate buffer, pH 5.5 (165 mg sodium acetate/10 ml) was then prepared.To enable formation of a stable thioether bond between the protein andliposome, a 10 μl aliquot of the freshly prepared DTT solution was addedto a mixture of the SPDP-labeled Mab and maleimide-derivatized liposomesat pH 6.5 which was incubated overnight at 25° C.

The method described above resulted in the introduction of 1 to 6 SHgroups per antibody as determined by the 5,5′ Dithio-(bis(nitrobenzoicacid) (DTNB) method (Deakin et al., Biochem. J, 89:296, 1963). In thismethod, unreacted —SH groups on the protein are blocked with DTNB whileunreacted maleimide groups on the liposome are blocked with N-ethylmaleimide. The number of thio groups introduced into the protein isassessed by monitoring the absorbance of free thiopyridone groups at 343nm that are liberated as the labeling proceeds.

The substrate-loaded liposomes were sensitized on the outer surface byreaction with SH-containing antibody for 2-3 hours at 25° C. Liposomeswere then applied to a Sepharose 6FF (Pharmacia) size exclusion columnequilibrated in storage buffer containing 2-10 mg/ml BSA, 1.3% glycerol,0.0005% dimethyl sulfoxide (DMSO), 0.74% EDTA. Liposomes were diluted instorage buffer that was supplemented with a 3:1 mixture ofsucrose:trehalose such that the final concentration of lipid in thesuspension was between 0.05 to 0.40 μmole/ml and the final concentrationof total sugar was between 5 and 10 mg/ml. This was then lyophilizedinto either polyester spunlace fabric or non-woven rayon.

To determine substrate release kinetics from the liposomes in a lateralflow one-step device, the lyophilized materials were cut and assembledas intermediate zones into a device in a manner described inInternational Publication No. WO92/12428. Capture zone membranes werespotted with anti-hCG polyclonal antibody (1-5 mg/ml) conjugated to thecorresponding enzyme or substrate releasing factor. The substratereleasing factor was either phospholipase (1-10 mg/ml), complementcomponent C₁q (0.1-4 mg/ml) or non-ionic polymeric detergents (0.1-0.4%)such as polyoxyethylene alcohols, polyoxyethylene-p-t-octylphenols,polyoxyethylenenonyl phenols, polyoxyethylene sorbitol esters,polyoxypropylene-polyoxyethylene esters of the Triton WR series.Phospholipase may also be attached to the capture antibody directly.

Substrate release from liposomes was studied by applying urine sampleswith or without hCG to the sample receiving zone and measuring enzymeproduct accumulation in the capture zone for insoluble products or inthe absorbent pad for soluble products. In the case of a coloredinsoluble product, the optical density was measured using a Umax 6SEflatbed scanner calibrated with a Kodak paper gray scale. In the case ofwater-soluble products which would precipitate out around the captureline, a densitometer (BioRad Laboratories, Hercules, Calif.) was usedwhich was also calibrated with a Kodak paper gray scale. In both cases,a correlation was observed between gray scale optical density units andanalyte concentration that can be used to estimate the analyte level inan unknown specimen.

To assess substrate release from liposomes in a flow-through assayformat, the unsupported media containing lyophilized liposomes were cutinto 2 cm diameter circles and assembled as a top layer of the device.Capture membranes were prepared similarly as described for the lateralflow device. Substrate release from liposomes was studied by applyingurine samples and performing similar measurements as for a lateral flowdevice.

EXAMPLE 3 Preparation of Biodegradable Substrate-Loaded Particles

Biodegradable synthetic--polyalkylcyanoacrylate (PECA) polymer monosizecolloids able to sorb internally sufficient quantities of desirableenzyme substrates were used as carriers for controlled delayed substratedelivery. The general procedure for the polymerization of colloidalparticles for sustained drug delivery is described in detail by Cicek etal. (in Biodegradable Polymeric Biomaterials, E. Piskin, Ed., MarcelDekker, N.Y., 1993, hereby incorporated by reference). This procedurewas modified to produce particles loaded with an appropriate enzymesubstrate in a desired particle size range for either lateral orflow-through assembly and exhibiting adequate degradation kinetics for aparticular diagnostic assay format. To this end, the concentrations ofthe components used in the polymerization process were varied asdescribed herein.

The PECA particles were prepared by polymerization of monomers of2-ethylcyanoacrylate (ECA) in an acidic aqueous medium containing thedesired enzyme substrate. Polymerizations were performed using acopolymer of polyethylene oxide (PEO)/polypropylene oxide (PPO) and arelatively high molecular weight (10,000-50,000) dextran. The dispersionmedium consisted of an aqueous solution of hydrochloric acid (HCI) andphosphoric acid (H₃PO₄). All components were of analytical reagent gradeand PEO/PPO copolymer was identical or similar to that commerciallyavailable as F-88 Pluronic Polyol from BASF.

To produce monosize PECA enzyme substrate-loaded particles, the ECA,PEO/PPO dextran, HCl, H₃PO₄ and enzyme substrate concentrations werevaried between 0.1-12.0% (v/v), 0.2-40 mg/ml, 0.3-100 mg/ml, 0.1-1.5 N,0.1-15% (v/v), and 0.2-10 mg/ml, respectively. In a typicalpolymerization process, 1 ml of ECA was added dropwise to 100 ml ofvigorously stirred (i.e., ≧1,000 rpm) solution containing PEO/PPO,dextran, phosphoric acid, enzyme substrate and hydrochloric acid, at theconcentration ranges indicated above. The sealed vials containing thereaction mixture were stirred at ambient temperature for between 6 and64 hours and the resultant reaction mixture was processed in a mannerconventional for the manufacture of monosize latex particles. Following2-3 routine washes of the particles with an acidic (<pH 4.0) weak buffersolution, the particles were sized to produce colloids loaded with anenzyme substrate within the desired diameter size. Subsequently, thesubstrate-loaded PECA particles were diluted in wash buffer to 0.1% to5% solids. The suspension was poured onto a nonbibulous support,abruptly frozen and lyophilized. Alternatively, the supports containingsubstrate-loaded particles were air dried overnight in a 45° C. oven orfor 24 hours over P₂O₅ in vacuo.

To determine substrate release kinetics from the particles in a lateralflow one-step device, the resulting materials were cut into 10×4 mmrectangles and assembled as intermediate zones into a device in a mannerdescribed in published PCT Application WO92/12428, which is herebyincorporated by reference. Capture zone membranes were prepared usingappropriate enzyme for the particular substrate-loaded PECA particles.Substrate release from PECA particles in buffer solutions of differentpH (1.5, 5.0, 6.0, 7.0, 7.4, 8.0, 9.6, 10.5) was studied by applyingbuffers to the sample receiving zone and measuring color accumulation inthe enzyme capture zone for insoluble products or intensity of the colorin the absorbent pad for soluble products.

To determine substrate release kinetics from the particles in aflow-through format, the unsupported media containing substrate-loadedPECA particles were cut into 2 cm diameter circles and assembled as atop layer of the device. Substrate release was studied by applyingbuffer solutions as described above and performing similar measurementsas for a lateral flow one-step device. The following observations weremade: (1) The substrate-loaded particles obtained with higher amounts ofPEO/PPO released substrate faster; (2) Decreasing HCl concentration inthe dispersion medium resulted in faster substrate release; and (3)Particles degraded faster, releasing more efficiently with increasing pHof the sample buffer medium.

4. Enzyme product chemical capture

In this embodiment, the enzyme product binds specifically to the supportwhich is coated or chemically derivatized with a functional chemicalgroup. Thus, the enzyme product is captured specifically versus simplyprecipitating at the test line. This technique is schematicallydiagrammed in FIG. 3. The capture antibody 42 binds to theanalyte-antibody-enzyme complex 44 at the test line 46. Enzyme substrate(S) is then converted to product (P) which binds to functional groups(R) at the test line 46 or downstream to form a chemical bond withlocalization and registration of enzyme by color or other means (i.e.,fluorometric, radiolabel, etc.).

EXAMPLE 4 Specific Chemical Capture of Enzyme Product

Compounds capable of reacting specifically with enzyme products wereincorporated into the capture zone. For the substrates of alkalinephosphatase, galactosidase or glucuronidase, diazotized amines wereincorporated into the capture zone. Examples of such compounds includediazonium salts (“fast salts”) and diazotized derivatives of polyamines,or natural polypeptides, such as albumin, immunoglobulins and the like.After enzyme-mediated formation of phenolic products, the diazotizedamines react in situ therewith to form stable colored azophenolcompounds. Alternatively, the chemical compounds were incorporateddownstream from the capture zone [i.e., either in the absorbent pad orin additional intermediate zone(s)] to allow detection of enzymeproducts away from the capture zone.

5. Delay zone dissolution for delivery of substrate and/or conjugate

This lateral flow embodiment uses separate paths that employtime-delayed dissolution of barrier zones for delivery of substrateand/or conjugate reagents to the test area. This facilitates earlyrelease and migration of antibody enzyme label (as conjugate), whichfacilitates delayed release of substrate for catalysis. Barrier 1dissolves first to release enzyme-antibody conjugate, while barrier 2dissolves later to release substrate. The enzyme product precipitates atthe test line or is measured downstream.

EXAMPLE 5

Delayed enzyme substrate delivery using hydrogel barrier zones

Referring to FIG. 4, to construct delayed enzyme substrate deliveryusing structural hydrogel barrier zones, the desired enzyme substratefor the corresponding enzyme was dissolved in the pH activity optimumbuffer at 0.2-3 mg/ml and supplemented with 10 mg/ml BSA prepared in thesame buffer. The enzyme substrate mixture was then lyophilized in anonbibulous support 50 such as polyester or polyacrylic spunlace fabric.A printed hydrophobic polyurethane barrier line 52 separates nonbibuloussupport 48 (no substrate) from nonbibulous support 50 (containingsubstrate). The enzyme-antibody label pad 58 was prepared by pouring theenzyme-antibody conjugate (5-50 μg/ml) supplemented with the appropriateactivators and stabilizers onto a similar support followed bylyophilization. Capture zone nitrocellulose membranes 50 were spottedwith anti-hCG polyclonal antibody at 1 to 5 mg/ml and blocked with 10mg/ml BSA or 0.2-2% (w/v) polyvinyl alcohol. Fluid communication bridges64 permit fluid flow between the sample pad-label pad and labelpad-capture zone.

In order to determine delayed enzyme substrate delivery in a lateralflow “one-step” device, the materials described above were cut andassembled as consecutive intermediate zones into a device except that a3-10 mm gap 54 was created between enzyme substrate pad 50 andenzyme-antibody label pad 58. Subsequently, a strip of Hypan TAU92(Taupan) molecular hydrogel sponge was laid down against the edge of theenzyme substrate pad, leaving a gap between the enzyme-antibody labelpad.

Structural (molecular) hydrogel barrier sponges 56 were constructedusing Hypan polymers (Hymedix International, Inc., Dayton, N.J.) whichare hydrophilic acrylate derivatives with multi-block copolymerstructures of several sequences of amorphous units with pendanthydrophilic groups derived from acrylic acid (soft blocks) responsiblefor swelling (flexibility) and several sequences of organized,crystalline pendant polyacrylonitrile structures (hard blocks)responsible for mechanical properties. Particularly preferred polymersare highly swelling associative polymers known as Hypan TN (TransientNetwork) Hydrogels, whose hard blocks form reversible meltable transientclusters. Subsequently, substrate release was studied by simultaneouslyapplying urine or serum samples with or without various levels of hCG tozones 48 and 50 of a sample pad divided by a barrier zone 52 andmeasuring enzyme product accumulation in the capture zone 60 forinsoluble products or in the absorbent pad 62 for soluble products.

As shown in FIG. 5, sample is added concurrently to both zones 48 and 50of a sample-pad that has been divided by a barrier zone 52 that has beenprinted onto Sontara spunlace fabric. Zone 50 is loaded with enzymesubstrate that has been lyophilized. Due to hydrophobic barrier 52, halfof the sample volume sits in zone 50 until the hydrogel barrier zone 56imbibes enough fluid to expand and close the gap 54 separating zone 50from the label pad 58. Meanwhile, the fluid in zone 48 has made progressforward such that the enzyme-antibody label has been picked up and theimmune complex formed at the capture line 61 within the nitrocellulosemembrane 60. At a later time, the substrate picked up in zone 50 arrivesat the capture line 61 which now has immobilized antibody associatedtherewith. The delay imposed by the expansion time required by Hypan toclose the gap has allowed time for bound/free separation of uncomplexedantibody and analyte to occur before arrival of the substrate.

EXAMPLE 6 Sequential Delayed Enzyme-Antibody and Enzyme SubstrateRelease Using Hydrogel Barrier Zones

Referring to FIG. 5, in a variation of Example 5, a 3-10 mm gap 54 wasalso created between the enzyme-antibody zone 58 and the capture zone60, and a strip of Hypan sponge 66 was laid down against the edge of theenzyme-antibody label pad 58, leaving a gap 54 between the capture zone60 and the label pad 58. To achieve sequential delayed release ofenzyme-antibody label after initial incubation with the sample (toincrease immunological efficiency), followed by delayed enzyme substraterelease from the enzyme substrate zone, different widths of sponge wereused to facilitate desired swelling time.

The performance of the device was tested by simultaneously applyingurine or serum samples without or with various levels of hCG to theenzyme substrate zone 50 and the enzyme-antibody label zone 58, andmeasuring enzyme product accumulation in the capture zone 60 forinsoluble enzyme products or in the absorbent pad 62 for solubleproducts.

The Hypan sponge 66 increases the dwell time of the sample analytewithin the label pad 58 to promote increased immunological efficiency.As sponge 66 swells, it closes the gap between the label pad 58 andcapture zone 60, permitting fluid communication between these sections.Hypan sponge 56 closes somewhat later such that delivery of the enzymesubstrate occurs after bound/free separation of surplus enzyme conjugateis essentially complete.

EXAMPLE 7 Sequential Delayed Enzyme-Antibody and Enzyme SubstrateRelease Using Enterosoluble Coating Barrier Zones

In a variation of the example just described, the enterosolublemethacrylic acid copolymer coatings similar to those used for solidpharmaceutical dosage (Eudragit S100; Rohm Tech., Inc., Malden, Mass.)were used to achieve sequential release of enzyme-antibody labelfollowed by enzyme substrate. For example, to achieve sequential delayedrelease of alkaline phosphatase (ALP)-labeled anti-hCG monoclonalantibody conjugate followed by the ALP substrate, the conjugate (0.5-10μg/ml) was prepared in 50 mM Tris-HCl buffer (pH 8.5) containing 5 mg/mlBSA, 1 mM MgCl₂ and 0.1 mM ZnSO₄, then lyophilized into a nonbibuloussupport as described above. The desired ALP substrate (i.e.,3-indoxylphosphate) was dissolved at 0.4-3 mg/ml in 0.1 M2-amino-2-methyl-1-propanol (AMP) buffer (pH 10.5) and lyophilized intoa nonbibulous support as described above. Capture zone membranes werespotted with anti-hCG monoclonal antibody at 1-5 mg/ml and blocked with10 mg/ml BSA or 0.2-2% (w/v) polyvinyl alcohol.

Subsequently, the materials were cut and assembled as consecutiveintermediate zones into a lateral flow one-step device as shown in FIG.6, except that 2-5 mm gaps were created between the ALP substrate padand the ALP-monoclonal antibody label pad, as well as between the labelpad and capture zone. In order to create the desired barrier zones, twoanionic copolymer mixtures based on methacrylic acid (MAA) andmethylmethacrylate (MMA) were prepared in methanol at 3-15% (w/v).Copolymer A comprised a MAA to MMA ratio of 12:88 and Copolymer Bcomprised a MAA to MMA ratio of 3:97. Copolymers A and B were driedseparately in nonbibulous supports 68 and 70, respectively.Subsequently, the gap between the ALP substrate pad and theALP-monoclonal antibody (MAb) label pad (see FIG. 5) was closed byattaching--rectangles of support 70 containing Copolymer B with a 1 mmoverlap on both sides. Similarly, the gap between the label pad andcapture zone (see FIG. 5) was closed using support 68 containingCopolymer A.

Copolymer B, having the lower ratio of MAA to MMA, will dissolve moreslowly than copolymer A and will enable delivery of the enzyme substrateto the capture zone later than enzyme-antibody conjugate. Due to thegenerally high pH (8-9.5) required by the ALP enzyme reaction, themethacrylic acid units in the copolymer ionize to bring about breakdownand dissolution of the copolymer. Copolymer A, with its higher mole %MAA, dissolves relatively quickly, allowing earlier delivery ofenzyme-antibody conjugate to the capture line, but increases the dwelltime of the analyte within the label pad sufficiently to enhanceimmunological efficiency.

Performance of the device was assessed by simultaneously applying urinesamples, with or without hCG, to the ALP substrate pad and ALP-MAb labelpad and performing similar measurements as described above.

EXAMPLE 8 Sequential Delayed Release of Enzyme-Antibody Label and EnzymeSubstrate Using Biodegradable Phospholipid Barrier Zones

In a variation of Example 7, biodegradable phospholipid barriers wereused to achieve sequential release of antibody-enzyme label followed byenzyme substrate in the lateral flow one-step device. ALP-MAb label padand ALP substrate pad were prepared as described in Example 7, exceptthat the nonbibulous supports were supplemented with various amounts ofphospholipase (1-20 μg/ml), with the concentration in the substrate padlower than that in the label pad to allow dissolution of theenzyme-antibody barrier zone prior to dissolution of the substratebarrier zone. Subsequently, the pads and capture zone were assembledinto a lateral flow one-step device with 2-5 mm gaps left between thesubstrate and label pad, as well as between the label pad and capturezone. To create the desired barrier zones, liposomes were prepared asdescribed in Example 2 except that enzyme substrate was not included andliposomes were not extruded through the membranes. Liposomes were driedin a nonbibulous support and gaps were closed using the liposomesupport.

Performance of the device was assessed by simultaneously applying urinesamples (with or without hCG) to the ALP substrate pad and ALP-MAb labelpad, and performing similar measurements as described in the previousexample.

6. Delayed release of enzyme substrate using protected enzyme substrate

This embodiment encompasses multi-enzyme systems wherein thetime-dependent production of the end product of the first enzymereaction is the substrate for the enzyme-antibody conjugate reaction.Referring to EIA device 72 shown in FIG. 7, substrate (S1) for the firstenzyme (E1) is immobilized in excess in a sample pad 74. Afterimmunological separation upon lateral flow of a solution containing ananalyte (X), the time-dependent catalysis of the substrate for theenzyme-antibody complex (E2-Ab2) begins with E1 acting on S1 to producesubstrate 2 (S2) in label pad 76. When the concentration of S2 reachesan effective threshold concentration for E2 to become active, theproduct (P) begins to form in the capture zone 78. By this time,sufficient delay has occurred so that most or all of the target analytehas been recognized and captured at the test line 80. The product (P) isthen directly quantified by absorbance as a colored product,electrochemically as an electroactive substance, fluorometrically,luminescently or is free to participate in a secondary chemical reactionso as to make possible one of these modalities of detection either as acaptured material or measured downstream as a soluble material. Unboundanalyte and antibodies flow to absorbent zone 82. A specific example ofthis EIA is described in Example 9.

EXAMPLE 9

In this example, referring to FIG. 7, E2-Ab2=β-D-galactosidase-anti-hCG;E1=ALP; S1=o-nitrophenyl-β-D-galactopyranoside-6-phosphate;S2=o-nitrophenyl-β-D-galactopyranoside; Ab1=goat-anti-hCG;P=o-nitrophenol. The conjugate of E. coli β-D-galactosidase and ananti-hCG MAb was prepared in 50 mM HEPES, pH 7.5 containing 1 mM MgCl₂and diluted to 0.5-10 μg/ml in the same buffer supplemented with 5 mg/mlBSA (the conjugate diluent). ALP was added to the conjugate solution toa final concentration of 10 μg/ml to 1 mg/ml, and the final mixture waslyophilized into a nonbibulous support as described above (the samplepad). O-nitrophenyl-β-D-galactopyranoside-6-phosphate cyclohexylammoniumsalt (S1) (Sigma) was dissolved at 0.1-1.5 mg/ml in the conjugatediluent and lyophilized into a nonbibulous support as described above(the label pad). The capture zone was prepared as previously described.Subsequently, the just-described materials were cut and assembled asconsecutive intermediate zones into a lateral flow one-step device.

Performance of the device and delayed release of enzyme substrate wasassessed by applying urine samples (with or without hCG) to the labelpad and performing similar measurements as previously described for alateral flow one-step device. The reaction scheme is summarized in FIG.8 in which S1=O-nitrophenyl-βD-galactopyranoside-6-phosphate;S2=O-nitroophenyl-β-D-galactopyranoside and P1=O-nitrophenol. Theproduct was measured colorimetrically by absorbance.

Alternative “protected” substrates for the β-D-galactosidase-E2conjugate and corresponding enzymes E1 are shown in FIG. 9. Table 2shows alternative “protected” substrates for the E2-Ab conjugate whenthe β-D-galactose substrate core is replaced with other sugars, as wellas the corresponding enzymes E2. FIG. 10 shows “protected” substratesfor urease as E1 in the E1-Ab conjugate.

In each case in Table 2 below, the sugar group in the left column wouldbe protected by the presence of a phosphate group or other protectinggroup as indicated in FIG. 9. The nitrophenyl, bromophenyl,chlorophenyl, methoxyphenyl or aminophenyl groups are removed by thecorresponding enzyme in the right column and the product quantified bythe appropriate modality.

TABLE 2 Substitute for β-D-galactase Enz₂ 2-acetamido-2-deoxy-β-D-N-acetyl glucosaminidase (EC glucosaminide 3.2.1.52) α-L-arabinofuranoseα-L-arabinofuranosidase III β-D-cellobiose exglucanaseN,N′-diacetyl-β-D-chitobiose chitobiosidase α-L-fucopyranoseα-L-fucosidase (EC 3.2.1.51) β-D-fucopyranose β-D-glycosidaseα-D-galactose α-galactosidase (EC 3.2.1.23) β-D-glucose β-glucosidase(EC 3.2.1.21) α-D-glucose glucansucrase β-D-glucopyranosiduronic acidβ-D-glucoronidase α-D-maltoheptose α-amylase (EC 3.2.1.1)α-D-maltohexose α-amylase (α or β)-mannopyranose (α or β)-mannosidase

It should be noted that the present invention is not limited to onlythose embodiments described in the Detailed Description. Any embodimentwhich retains the spirit of the present invention should be consideredto be within its scope. However, the invention is only limited by thescope of the following claims.

What is claimed is:
 1. A lateral flow enzyme immunoassay device,comprising: a sample pad comprising a first lane and a second laneseparated by a hydrophobic barrier, wherein said first lane contains anenzyme substrate and said second lane contains an enzyme-antibodyconjugate having affinity for an analyte; a capture zone separate fromsaid sample pad, which is in fluid communication with said first laneand said second lane of said sample pad prior to initial application ofa sample, said capture zone having a capture antibody incorporatedtherein having affinity for said analyte; and an absorbent zone in fluidcommunication with said capture zone; wherein said second lanetransports a fluid at a faster rate than said first lane.
 2. Theimmunoassay device of claim 1, wherein said analyte is selected from thegroup consisting of hormones, enzymes, lipoproteins, bacterial or viralantigens, immunoglobulins, lymphokines, cytokines, drugs and solublecancer antigens.
 3. The immunoassay device of claim 1, wherein saidsample pad comprises high density polyethylene.